ORIGINAL PAPER

Involvement of Lysophosphatidic Acid, Sphingosine 1-Phosphateand Ceramide 1-Phosphate in the Metabolization of PhosphatidicAcid by Lipid Phosphate Phosphatases in Bovine Rod OuterSegments

Susana J. Pasquare Æ Gabriela A. Salvador ÆNorma Maria Giusto

Accepted: 12 December 2008 / Published online: 21 February 2008

� Springer Science+Business Media, LLC 2008

Abstract The aim of the present research was to evaluate

the generation of [2-3H]diacylglycerol ([2-3H]DAG) from

[2-3H]-Phosphatidic acid ([2-3H]PA) by lipid phosphate

phosphatases (LPPs) at different concentrations of lyso-

phosphatidic acid (LPA), sphingosine 1-phosphate (S1P),

and ceramide 1-phosphate (C1P) in purified ROS obtained

from dark-adapted retinas (DROS) or light-adapted retinas

(BLROS) as well as in ROS membrane preparations

depleted of soluble and peripheral proteins. Western blot

analysis revealed the presence of LPP3 exclusively in all

membrane preparations. Immunoblots of entire ROS and

depleted ROS did not show dark–light differences in LPP3

levels. LPPs activities were diminished by 53% in BLROS

with respect to DROS. The major competitive effect on PA

hydrolysis was exerted by LPA and S1P in DROS and by

C1P in BLROS. LPPs activities in depleted ROS were

similar to the activity observed in entire DROS and

BLROS, respectively. LPA, S1P and C1P competed at

different extent in depleted DROS and BLROS. Sphingo-

sine and ceramide inhibited LPPs activities in entire and

depleted DROS. Ceramide also inhibited LPPs activities in

entire and in depleted BLROS. Our findings are indicative

of a different degree of competition between PA and LPA,

S1P and C1P by LPPs depending on the illumination state

of the retina.

Keywords Rod outer segments � Lipid phosphate

phosphatases � Phosphatidic acid � Diacylglycerol

Abbreviations

C1P Ceramide 1-phosphate

DAG Diacylglycerol

DTT Dithiotreitol

EDTA Ethylenediaminetetraacetic acid

EGTA Ethylene glycol bis (b-aminoethyl ether)-

N,N,N0,N0-tetra acetic acid

LPA Lysophosphatidic acid

LPPs Lipid phosphate phosphatases

NEM N-ethylmaleimide

PA Phosphatidic acid

PAP2 NEM-insensitive phosphatidate phosphohidrolase

PC Phosphatidylcholine

PMSF Phenylmethylsulfonylfluoride

S1P Sphingosine 1-phosphate

TLC Thin layer chromatography

Introduction

Lipid phosphate monoesters, including phosphatidic acid

(PA), lysophosphatidic acid (LPA), sphingosine 1-phos-

phate (S1P) and ceramide 1-phosphate (C1P), are

intermediaries in phospho- and sphyngolipid biosynthesis

and they also play important roles in intra- and extracel-

lular signaling. Dephosphorylation of these lipids

terminates their signaling actions and generates products

with additional biological activities or metabolic fates [1].

The key enzymes responsible for the dephosphorylation of

these lipid phosphate substrates are termed lipid phosphate

phosphatases (LPPs). The latter display isoforms and cell

specific localization patterns which are distributed between

endomembrane compartments and the plasma membrane.

The role of LPPs in intracellular lipid metabolism and in

the regulation of both intra- and extracellular signaling

S. J. Pasquare � G. A. Salvador � N. M. Giusto (&)

Instituto de Investigaciones Bioquımicas de Bahıa Blanca,

Universidad Nacional del Sur and Consejo Nacional de

Investigaciones Cientıficas y Tecnicas (CONICET), C.C. 857,

B8000FWB Bahia Blanca, Buenos Aires, Argentina

e-mail: ngiusto@criba.edu.ar

123

Neurochem Res (2008) 33:1205–1215

DOI 10.1007/s11064-007-9569-5

pathways that control different cellular functions have been

described [2–5].

Rod outer segments (ROS) have the ability to adapt the

sensitivity and speed of their responses to ever changing

conditions of ambient illumination. Recent evidence has

demonstrated that a major contributor to this adaptation is

the light-driven translocation of key signaling proteins into

and out of ROS, which constitute the cellular place where

phototransduction occurs [6]. It has also been reported that

transducin, arrestin and recoverin [7–10] are proteins

involved in this mechanism. Previous studies revealed the

presence of phosphatidate phosphohydrolase 2 (PAP2,

renamed as LPPs) and its regulation in isolated ROS from

bovine retina [11–14]. In this work, the generation of

[2-3H]DAG from [2-3H]PA was analyzed either in the

absence or in the presence of LPA, S1P or C1P, all of

which are alternative substrates for LPPs. The presence of

LPP3 isoform in isolated ROS was also determined by

western blot.

It has been extensively reported that the activity of enzymes

involved in ROS phospholipid turnover such as phospholipase

C [15, 16], phospholipase A2 [17], phosphatidylethanolamine

N-methyltransferase [18], diacylglycerol kinase [19], PAP2

[14], phosphoinositide-3-kinase [20, 21], and phospholipase

D [22] is modulated by light. This fact and the evidence that

light induces a redistribution of proteins involved in photo-

transduction open an attractive field for the study of PA

hydrolysis by LPPs under illumination conditions. We have

also characterized LPP activities in the presence of [2-3H]PA

co-incubated with LPA, or S1P or C1P in ROS prepared from

dark-adapted retinas (DROS) as well as in ROS prepared

from light-adapted retinas (BLROS). Our results provide

evidence that PA is differently hydrolyzed by LPPs in the

presence of LPA, S1P, and C1P. Light participation in the

competition of LPA, S1P and C1P on PA metabolization is

also demonstrated.

Experimental Procedure

Materials

[2-3H] Glycerol (200 mCi/mmol) was obtained from New

England Nuclear-Dupont, Boston, MA. Sphingosine 1-

phosphate, ceramide 1-phosphate from bovine brain, oleoyl-

L-a-lysophosphatidic acid, D-sphingosine, and non-hydroxy

fatty acid ceramide from bovine brain were obtained from

Sigma-Aldrich, St. Louis, MO, USA. Monoclonal antibody

against rhodopsin, Rho4D2, was generously supplied by

Robert Molday from the University of British Columbia,

Vancouver, Canada. The secondary antibody used by the

rhodopsin immunoblots was HRP-conjugated anti-mouse.

Anti-PAP2B (anti-LPP3) was from Upsate biotechnology

(catalog number 07-142). The secondary antibody used for

anti-PAP2b detection was HRP-conjugated anti-rabbit. All

the other chemicals were of the highest purity available.

Dark and Light Adaptation Procedure

Bovine eyes were obtained from a local abattoir, placed on

ice within 10 min of the animal’s death, and they were kept

in darkness for 2 h. Retinas were dissected from the eyes

after dark- or light adaptation. Dark-adapted bovine ROS

(DROS) and light-adapted bovine ROS (LROS) were pre-

pared under dim red light from DROS. The bleaching

process was carried out after removing the cornea and the

lens from the eyeballs and by exposing them to light

(300 W) at 30 cm for 30 min at room temperature [20].

Controls were performed under the same conditions but

under darkness (DROS).

Rod Outer Segment Isolation

The subsequent procedures for ROS preparation were

conducted under dim red light for DROS and LROS, and

under room light for BLROS and they were carried out at

2–4�C. To isolate ROS, retinas were removed and shaken

twice in a 40% sucrose solution containing 1 mM MgCl2,

1 mM DTT, 0.1 mM PMSF, 1 lg/ml aprotinin, and 2 lg/

ml leupeptin in 70 mM sodium phosphate buffer (pH 7.2).

The remains of retinas were sedimented at 2,200g for

4 min and the supernatants containing ROS were diluted

1:2 with sucrose-free buffer and then centrifuged during

30 min at 35,300g. ROS were purified by a discontinuous

gradient of sucrose [23], yielding: (i) a ROS band (Band I)

retained at the 0.84/1.00 M density interface; (ii) a band

(Band II) retained at the 1.00/1.14 M density interface of

broken ROS contaminated with mitochondria and RIS, and

(iii) a pellet composed of non-ROS membranes. The purity

of ROS membrane preparations was monitored by electron

microscopy. Electron micrographs from purified ROS

(Band I) showed intact ROS with their typical structures

and no other membrane material was observed (data not

shown). The purity of Band I was also controlled by the

determination of the absorbance ratio at 278 and 500 nm of

solubilized membranes in 70 mM potassium phosphate

buffer (pH 7) containing 1% emulphogene. Values of

2.3 ± 0.2 were typically obtained for this ratio. In addition,

the purity of membranes was checked by sodium dodecyl

sulfate–polyacrylamide gel electrophoresis [24]. Even in

overloaded gels (80 lg of ROS protein), rhodopsin com-

prised 85–90% of photoreceptor integral membrane

proteins. Moreover, thin-layer chromatography of

1206 Neurochem Res (2008) 33:1205–1215

123

photoreceptor membrane lipids in overloaded plates

showed no cardiolipin, thus suggesting non-detectable

contamination with mitochondria. Instead, an enrichment

of long-chain polyunsaturated fatty acids esterified to di-

polyunsaturated molecular species of PC was observed.

This enrichment, which is characteristic of bovine ROS

[25]. Purity of ROS populations, particularly batches, was

determined by assaying marker enzymes activities in all

fractions of the gradients. NADPH-cytochrome c-reductase

(microsomal marker) and cytochrome c-oxidase (mito-

chondrial marker) activities were measured in Band I, Band

II and pellet of the gradient. Cytochrome c-oxidase was

more enriched in Band II and pellet whereas the micro-

somal marker activity was very low in Band I and

evidenced the highest activity in the pellet. These results

led us to confirm that Band I (purified ROS) contamination

with microsomes or mitochondria was lower than 5% [26,

27].

Soluble and Peripheral Protein Extraction from ROS

with Low Ionic Strength Buffer

ROS pellets from DROS or BLROS were resuspended (1 mg

protein per ml) in low ionic strength buffer prepared with

5 mM Tris–HCl (pH 7.4), containing 0.5 mM MgCl2, 1 mM

DTT, 0.1 mM PMSF, 1 lg/ml aprotinin, 2 lg/ml leupeptin

and 1 lg/ml of pepstatin, followed by 15 passages through a

G25 5/8 needle. The membrane suspensions were centri-

fuged at 35,300g for 30 min. The supernatants were removed

and centrifuged again in order to ensure that all particulate

material was sedimented and membrane pellets were

extensively washed. Both membranes and the clear super-

natants were analyzed to determine the polypeptide

composition, the presence of LPP3 and the activity of LPPs.

Preparation of Radioactive 1,2-diacyl-sn-glycerol-3-

phosphate

Radioactive phosphatidic acid was obtained by the action

of phospholipase D on [2-3H]-phosphatidylcholine [28].

[2-3H]-phosphatidylcholine was synthesized from bovine

retinas incubated with [2-3H] glycerol (200 mCi/mmol) as

previously described [11]. Lipids were extracted from the

tissue following Folch et al. procedure [29]. [2-3H]-Phos-

phatidylcholine was isolated by mono-dimensional TLC

and eluted there from [30]. [2-3H]PA, the hydrolysis

product of [2-3H]-PC, was purified by one-dimensional

TLC on silica gel H developed with chloroform/methanol/

acetic acid/acetone/water (9:3:3:12:1.5, v/v). The substrate

was eluted from silica gel with neutral solvents to avoid the

formation of lysophosphatidic acid. It was subsequently

converted into free acid by washing it twice using an upper

phase containing 0.1 M sulfuric acid and then an upper

phase containing water. Radioactivity and phosphorous

content [31] were measured to determine specific radio-

activity. [2-3H]PA with a specific radioactivity of 0.1–

0.2 lCi/lmol was obtained.

Determination of LPPs Activities

For the determination of LPPs activities the assay contained

50 mM Tris–maleate buffer, pH 6.5, 1 mM DTT, 1 mM

EDTA plus 1 mM ethylene glycol-bis(b-aminoethylether

N,N,N0,N-tetraacetic acid (EGTA), 4.2 mM NEM and

100 lg of ROS membrane proteins in a volume of 200 ll.

The reaction was started by the addition of 100 lM of

[2-3H]-phosphatidate/Triton X-100 mixed micelles in a

constant 1:50 molar ratio of lipid to Triton X-100 [32].

[2-3H]-phosphatidate was dried under a stream of nitrogen

and resuspended in 30 ll of buffer assay containing Triton

X-100, this aqueous microdispersion was sonicated in a

sonication tip until clarity. The effect of the alternative

substrates on PA metabolization by LPP activities, was

evaluated using 100 lM [2-3H]PA/Triton X-100 mixed

micelles in the presence of LPA, S1P or C1P (previously

resuspended in the buffer assay containing Triton X-100) at

the indicated concentrations [32, 33]. Sphingosine and cer-

amide were solubilized in 0.1% of dimethyl sulfoxide

(DMSO) as vehicle; the results obtained were compared

against controls performed with the vehicle. The assays for

the determination of LPPs were conducted at 37�C for

30 min under dim red light (DROS), under room light

(LROS) or under 300 W light (BLROS). The enzymatic

assay was stopped by adding chloroform/methanol (2:1, v/

v). Blanks were prepared identically, except that membranes

were boiled for 5 min before being used. PA products gen-

erated by LPP activities were isolated and measured as

described below. The enzymatic activity was expressed as

the sum of nmol of [2-3H] diacylglycerol and [2-3H] mon-

oacylglycerol 9 (h mg protein)-1.

Extraction and Isolation of Lipids

Lipids were extracted with chloroform/methanol (2:1, v/v)

and washed with 0.2 volumes of CaCl2 (0.05%) [32]. Neutral

lipids were separated by gradient-thickness thin-layer chro-

matography on silica gel G [34] and developed with hexane/

diethyl ether/acetic acid (35:65:1, v/v). To separate mono-

acylglycerol (MAG) from phospholipids, the chromatogram

was rechromatographed up to the middle of the plate by using

hexane/diethyl ether/acetic acid (20:80:2.3, v/v) as devel-

oping solvent. Once the chromatogram was developed,

Neurochem Res (2008) 33:1205–1215 1207

123

[2-3H] PA and phospholipids were retained at the spotting

site. Lipids were visualized by exposure of the chromato-

grams to iodine vapors, scraped off the plate, and quantified

by liquid scintillation spectroscopy.

SDS–PAGE and Immunoblot Analysis

SDS–PAGE was performed using 7.5% or 10% gels

according to Laemmli [24]. Resolved proteins were trans-

ferred to immobilon P membranes using a Mini Trans-Blot

cell electro blotter (BIO-RAD Life Science Group, Cali-

fornia) for 1 h. Membranes were blocked overnight with

Tris-buffered saline (20 mM Tris–HCl, 300 mM NaCl) pH

7.5, containing 0.1% Tween 20 and 5% crystalline grade

bovine serum albumin (BSA). Incubations with primary

antiserum were performed at room temperature for 2–3 h.

Immunoreactions were detected by means of either horse-

radish peroxidase conjugated to goat antirabbit or goat anti-

mouse IgG followed by enhanced chemiluminescence

substrates (ECL; Amersham Biosciences, Inc.). In some

experiments, immunoblots were stripped by incubation in

200 mM Tris–HCl buffer, pH 6.7, containing 100 mM b-

mercaptoethanol, 2% sodium dodecyl sulfate (SDS) for 1 h

at 50�C with gentle agitation. Blots were reblocked with

5% BSA in Tris-buffered saline and probed as described

above. Immunoreactive bands were quantified using image

analysis software (Image J, a freely available application in

the public domain for image analysis and processing,

developed and maintained by Wayne Rasband at the

Research Services Branch, National Institute of Mental

Health, Bethesda, Maryland, USA).

Other Methods

Lipid phosphorus and protein were determined as described

elsewhere [31, 35].

Statistical Analysis

Statistical analysis was done using Student’s t-test with the

values representing the mean ± SD of the total number of

samples indicated in each figure legend.

Results

Hydrolysis of [2-3H]-phosphatidate by LPPs in DROS

and BLROS as a Function of LPA, S1P and C1P

Concentrations

The DAG formed by LPP activities is partially hydrolyzed

by diacylglycerol lipase (DAGL), yielding MAG. DAGL is

coupled to LPPs and it seems that these enzyme activities

are working in a sort of enzyme complex. Taking this into

account the DAG generation and its partial degradation by

DAGL immediately occurs. This fact has been extensively

described in our laboratory [11, 12]. For this reason the

most accurate from to express LPP activity is as the sum of

nmol of [2-3H] diacylglycerol and [2-3H] monoacylglyc-

erol 9 (h mg protein)-1.

Data presented in Fig. 1 show the rate of DAG forma-

tion from [2-3H]-phosphatidate (PA) in the presence of the

alternative substrates using two ROS populations: DROS

(square symbols) and BLROS (circle symbols). When the

light effect was evaluated on LPP activities using only

[2-3H]PA as substrate, an inhibition of 53% was observed

in BLROS with respect to DROS (Fig. 1). LPA and S1P

significantly decreased DAG (P \ 0.025) production from

[2-3H]PA in a concentration-dependent manner in DROS

(Fig. 1, square symbols). LPA diminished DAG formation

by 15% at concentrations ranging from 10 to 50 lM and by

29% at the highest concentration of LPA assayed (200 lM)

(Fig. 1A). MAG generation from LPA could also be cat-

alyzed by LPA phosphohydrolase. The presence of this

enzyme activity has not been described in isolated ROS.

Moreover, in our experiments with non-labeled LPA it is

not possible to evaluate the presence of this enzyme.

An important diminution in DAG production was

observed at low concentrations of S1P reaching 27% at

50 lM of S1P (Fig. 1B). C1P exerted an inhibitory effect

(30%) on DAG formation at concentrations higher than

100 lM (Fig. 1C).

In order to determine the effect of light (Fig. 1, circle

symbols) in the competition of LPA, S1P and C1P in PA

hydrolysis, LPP activities were also evaluated in BLROS in

the presence of increasing concentrations of the alternative

substrates for the enzyme. In BLROS a 22% decrease in

DAG formation was observed at 100 lM of LPA (Fig. 1

A). In BLROS PA dephosphorylation was diminished by

19% at 20 lM of S1P whereas at 100 lM of S1P, DAG

levels returned to basal levels (Fig. 1B). On the other hand,

C1P diminished DAG production at all the concentrations

assayed, being highest (41%) at 200 lM (Fig. 1C).

Hydrolysis of [2-3H]-phosphatidate by LPPs in LROS

and BLROS in the Presence of LPA, S1P and C1P

For the experiments aimed at the description of LPP

activities under different illumination conditions we used

three distinct ROS populations: (i) DROS: obtained from

dark-adapted retinas and purified under dim red light, (ii)

LROS: obtained from DROS and exposed to room light for

the enzyme assays, (iii) BLROS: obtained from light-

adapted retinas and purified under room light.

1208 Neurochem Res (2008) 33:1205–1215

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Figure 2 shows the percentage of LPPs activities in LROS

and BLROS with respect to the activities determined in

DROS (considered as 100%) in the absence or in the pres-

ence of 20 or 100 lM of LPA, S1P or C1P. LROS were

obtained, exposing DROS to room light. Under these

experimental conditions an inhibition of 24% in LPPs

activities with respect to DROS was observed (Fig. 2). LPA

and C1P did not modify LPPs activities from LROS

(Fig. 2A, C), whereas in the presence of S1P, LPPs activities

were stimulated by 13% (Fig. 2B). A differential effect of

alternative substrates in PA hydrolysis pattern was observed

between LROS and BLROS. In all cases, LPPs activities

were higher in LROS than in BLROS. Based on the results

shown in Fig. 2 a ratio between the percentages of LPPs

activities from LROS and BLROS was determined. This

ratio was 1.6 in the presence of PA as unique substrate, 1.3 in

the presence of LPA (20, 100 lM), 2.2 and 1.3 in the pres-

ence of either 20 or 100 lM of S1P, respectively, and it was 1

in the presence of 100 lM of C1P.

SDS–PAGE and Immunoblot Analysis

We determined the protein composition of entire DROS

and BLROS preparation and of DROS and BLROS subject

to successive washes with low ionic strength buffers. Low

ionic buffer treatment renders a ROS membrane prepara-

tion depleted of soluble and peripheral proteins. Both the

membrane and soluble fractions obtained there from were

used for SDS–PAGE analysis. The levels of arrestin were

higher in entire BLROS than in DROS (Fig. 3A). The

major polypeptides present in the ROS soluble fraction

(supernatant obtained after washing ROS preparations with

low ionic buffer) were a and b subunits of transducin (Ga,

Gb), arrestin and phosphodiesterase (PDE) (Fig. 3A). The

effect of GTP concentrations, and the rate of GTP hydro-

lysis participates in Gt translocation [6]. In our

experimental setup it seems that the initial concentration of

GTP at the moment of ROS isolation is enough for

allowing a significant light-mediated translocation of Gt.

Immunoblot analysis using RGD domain anti-phosphatidic

acid phosphatase 2b (anti-LPP3) revealed that this isoform

Fig. 1 Hydrolysis of [2-3H]-phosphatidate (PA) by LPPs in DROS

and BLROS as a function of lysophosphatidic acid (LPA), sphingo-

sine 1-phosphate (S1P) and ceramide 1-phosphate (C1P)

concentrations. LPPs activities were determined using, as an enzyme

source, either purified ROS from dark-adapted retinas (DROS) or

bleached ROS (BLROS) from DROS whose eye cups were exposed

to room light (300 W at 30 cm) for 30 min as specified in

Experimental Procedure. The effect of the alternative substrates on

PA hydrolysis by LPP activities, was evaluated using 100 lM

[2-3H]PA/Triton X-100 mixed micelles in the presence of LPA (A),

S1P (B) or C1P (C) at the indicated concentrations. The enzymatic

assay was made under dim red light for DROS or under 300 W light

for BLROS. Incubation products were subsequently extracted and

separated by gradient-thickness thin-layer chromatography and visu-

alized after exposure to iodine vapor. The bands corresponding to PA,

DAG and MAG were scraped and quantitated by liquid scintillation

spectroscopy. Results represent the mean ± SD of nine individual

samples. (*P \ 0.025)

b

Neurochem Res (2008) 33:1205–1215 1209

123

was present in the membrane fractions while it was absent

in the soluble fraction (data not shown). No light–dark

differences in LPP3 protein levels were observed either in

entire ROS or in depleted membranes (Fig. 3B). Opsin

immunoblots demonstrated that LPP3 co-localizes with the

membrane fraction and that the protein is absent in the

soluble fractions. Densitometry ratios (LPP3/opsin)

showed no differences in LPP3 levels when entire ROS and

depleted ROS were compared (Fig. 3C).

Hydrolysis of [2-3H]-phosphatidate by LPPs in DROS

and BLROS Depleted of Peripheral and Soluble

Proteins. Effect of LPA, S1P and C1P

In order to evaluate the effect of peripheral and soluble

proteins depletion on LPPs activities, enzyme assays were

carried out in membranes from DROS and BLROS washed

with low ionic strength buffer (Fig. 4). The same LPPs

activities and similar dark/light differences were observed

both in ROS and in the membranes treated with 5 mM

buffer (Fig. 4). No enzyme activity was detected in the

soluble fraction obtained from depleted DROS and BLROS

(data no shown). DAG generation from [3H]PA in depleted

DROS in the presence of LPA, S1P or C1P was 57%, 61%

and 86% with respect to the activity determined in the

presence of [3H]PA alone. In depleted BLROS membranes,

DAG generation in the presence of LPA and S1P was 64%

and 50% in the presence of C1P with respect to the activity

found in the presence of [3H]PA alone.

Hydrolysis of [2-3H]-phosphatidate by LPPs from

DROS and BLROS in the Presence of Sphingosine and

Ceramide

In order to determine if the effect of S1P and C1P on DAG

production was due either to their competitive character-

istics and/or to the sphingosine and ceramide, the

respective dephosphorylation products of S1P or C1P by

LPPs, sphingosine or ceramide were included in PA

hydrolysis assays. Sphingosine and ceramide were added

using DMSO as vehicle. ROS were preincubated with

sphingosine and ceramide but similar results were observed

when sphingosine and ceramide were co-incubated with

PA (data not shown). In entire DROS, sphingosine and

ceramide inhibited DAG production by a similar percent-

age (18%) at all concentrations assayed (Fig. 5A, B). In

entire BLROS sphingosine (100 lM) significantly inhib-

ited DAG production by 23% and ceramide (50, 100,

300 lM) yielded the same inhibition percentage as that of

Fig. 2 Hydrolysis of [2-3H]-phosphatidate by LPPs in LROS and

BLROS in the presence of LPA, S1P and C1P. BLROS were

obtained as was specified in Fig. 1. LROS were prepared following

the same procedure as that of DROS but the enzymatic assay was

made under room light. Results are expressed as the percentage of

LPP activities in LROS or BLROS with respect to DROS and they

represent the mean ± SD of nine individual samples. (**P \ 0.01)

1210 Neurochem Res (2008) 33:1205–1215

123

sphingosine (Fig. 5B). In ROS depleted of soluble and

peripheral proteins sphingosine and ceramide inhibited

DAG formation by 48% and 40% in DROS and BLROS,

respectively (Fig. 5C).

Discussion

The key enzyme LPPs are the main class of enzymes

responsible for the dephosphorylation in vitro of PA and of

the other phosphorylated substrates, such as LPA, S1P, and

C1P. Three mammalian LPP isoforms termed LPP1

(PAP2a), LPP2 (PAP2c), and LPP3 (PAP2b) have been

cloned [29]. These substrates are mutually competitive

with each other. In general, LPP1, LPP2, and LPP3 show

the major catalytic efficiency to LPA, PA, and S1P,

respectively [36, 37]. The primary function of LPPs is a

direct consequence of the dephosphorylation of their lipid

substrates, altering the balance of bioactive lipid mediators

[2, 38, 39]. PA and its dephosphorylated product, DAG,

have important functions in signaling and PA itself emer-

ges as a regulator of pleiotropic signaling responses [40].

ROS are responsible for the initial events of vision at

low light levels. Light modulation of lipid metabolism and

of a number of enzymes that generate second lipid mes-

sengers in ROS has been thoroughly studied [14, 16–19,

21, 22]. In addition, light-dependent membrane transloca-

tion of phototransduction proteins has been documented in

detail [7, 41, 42].

Arrestin, transducin and recoverin redistribute in rods in

response to bright light. Arrestin predominates at rod inner

segments from DROS shifting to ROS during light expo-

sure. Transducin a and b subunits shift in the opposite

Fig. 3 SDS–PAGE and immunoblot analysis. ROS depleted of

peripheral and soluble proteins were obtained from DROS or

BLROS. Supernatants were removed (soluble ROS fraction) and

membrane pellets (depleted ROS) were analyzed by SDS–PAGE and

immunoblot. (A) Commassie Blue-stained gel of entire ROS, ROS

soluble fraction and depleted ROS either from dark- or light-adapted

retinas. (B) Immunoblot of entire ROS, ROS soluble fraction and

depleted ROS from dark- or light-adapted retinas with RGD domain

anti-phosphatidic acid phosphatase 2b (anti-LPP3) (upper panel).

Immunoblots obtained with anti LPP3 antibodies were stripped and

reprobed with anti-opsin antibody. Each lane contains 30 lg of

protein. (C) LPP3/opsin ratios (expressed as arbitrary units) were

calculated after densitometry analysis of immunoblots

Fig. 4 Hydrolysis of [2-3H]-phosphatidate by LPPs in DROS and

BLROS depleted of peripheral and soluble proteins. Effect of LPA,

S1P and C1P. LPPs activities were assayed in depleted ROS obtained

from either dark- or light-adapted retinas. Membranes were incubated

with 100 lM of [2-3H]PA/Triton X-100 mixed micelle or with

[2-3H]PA in the presence of 100 lM of LPA, S1P or C1P as was

specified in Fig. 1. Results represent the mean ± SD of nine

individual samples (****P \ 0.001)

Neurochem Res (2008) 33:1205–1215 1211

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direction in response to light [7, 42] and a significant

reduction of recoverin is produced in ROS by light [10].

The differential LPP activity observed in DROS with

respect to LROS is only due to the light effect on ROS

obtained from DROS. In our study it was observed that

LPP activities are strongly inhibited in BLROS (50%). This

is indicative of the involvement of bleaching process and

light in LPP modulation. The light effect on LPP activities

in ROS obtained from DROS has been previously dem-

onstrated in our laboratory [14]. The important inhibition in

LPP activities exerted by light and the absence of differ-

ences in LPP3 levels between DROS and BLROS could be

related either to the absence or to the presence of a specific

protein affected by light-driven translocation. These find-

ings agree with our previous observations which

demonstrated that light inhibition of LPP activity in ROS is

a transducin-mediated mechanism [14]. PLD is inhibited

by light as it occurs with LPPs [22]. On the other hand, it

has been reported that diacylglyceride kinase (DAGK) [19]

is modulated by light in the opposite manner as it occurs

with LPPs and PLD. This could be indicating that PA and

DAG levels have physiological relevance in ROS under

illumination: i.e., under light conditions an increased

DAGK activity promotes a major PA availability, under

dark conditions an increase in PLD/PAP activities yields a

major DAG availability [14, 22].

We have previously described that experimental condi-

tions favoring PKC phosphorylation inhibits LPP in

purified ROS [13]. Light stimulates DAG generation by

PIP2-PLC activity [43] and thus may activate ROS PKC

and this could be at least one of the light-induced mecha-

nisms involved in LPPs modulation. Downstream signals

by the action of LPPs may play a key role in light photo-

receptor desensitization/adaptation. The functional

significance of light-modulated LPP activities in vertebrate

photoreceptors has not been fully elucidated to date.

However, a role for LPPs in Drosophila melanogaster

phototransduction has been reported. In Drosophila pho-

toreceptors, photoisomerized rhodopsin activates a Gq

protein releasing the a subunit, which in turn, activates

phospholipase C [44]. PLC activation leads to the opening

of two classes of Ca2+ permeable channels [45, 46]. In

Drosophila DAG and PA levels are regulated by the syn-

ergistic activity of DA6K and by the recently described

LPP gene called Lazaro [47].

In our study, DROS, LROS, and BLROS showed a

different PA hydrolysis pattern in the presence of LPA,

S1P, and C1P. S1P and LPA in DROS and LPA and C1P in

BLROS produced the highest competitive effect on PA

hydrolysis. Our study provides the first lines of evidence of

the existence of LPP3 (PAP 2b) in ROS. The results

Fig. 5 Hydrolysis of [2-3H]-phosphatidate by LPPs from DROS and

BLROS in the presence of sphingosine and ceramide. Entire ROS

obtained from dark- or light-adapted retinas were preincubated with

sphingosine (50, 100 and 300 lM) or ceramide (50, 100 and 300 lM)

for 10 min. Depleted ROS were incubated with sphingosine or

ceramide (300 lM). [2-3H]PA (100 lM)/Triton X-100 mixed

micelles was subsequently added and the enzymatic activity was

determined as specified in Fig. 1. Sphingosine and ceramide were

added in DMSO 0.1% and the vehicle always served as control.

Results represent the mean ± SD of nine individual samples.

(***P \ 0.005; ****P \ 0.001 with respect to entire DROS or entire

BLROS)

1212 Neurochem Res (2008) 33:1205–1215

123

observed in the presence of S1P reinforced the existence of

LPP3 in ROS [29]. In view of the results obtained with

LPA we cannot discard the presence of LPP1 and LPP2

besides LPP3 in purified ROS. LPP3 has been localized

with PLD in caveolin-enriched detergent-resistant micro-

domains (DRMs) where LPP3 metabolizes phospholipase

D2-derived PA [48, 49]. The presence of PLD activity [48]

and the existence of DRMs [50] in purified ROS, reinforce

a physiological role for LPP3 in this system.

Previous observations suggest that LPP3 protein regu-

lates cell–cell interactions and act as an ecto-enzyme [3,

51]. It was previously demonstrated that LPP from ROS is

mainly located in the disk membrane [11]. For this reason

ecto- LPP activity could provide lipid messengers in the

extracellular medium inside the ROS disks. The pattern

observed in the presence of S1P, mainly in DROS, could be

either a consequence of a competition with PA by LPPs or

it could be due to the formation of sphingosine from S1P,

which may inhibit PA hydrolysis. This possibility was

corroborated by our observations of the sphingosine effect

on DAG generation (Fig. 4). In this respect, it has been

reported that sphingosine not only inhibits DAG formation

but also stimulates PA formation, inhibiting LPPs and

stimulating PLD and DAGK [52, 53].

C1P is a potent inhibitor of protein phosphatases (PP)

and PPs have been implicated in the inhibition of LPPs in

isolated ROS [13, 54, 55]. Another possibility is that C1P

exerts a direct action on LPPs. It has been reported that

C1P is required for the activation and translocation of other

enzymes involved in lipid metabolism such as cytosolic

phospholipase A2 [56]. An inhibition in DAG production

from PA by ceramide was also observed. Ceramide itself is

an important second messenger in various stress responses

and there are several candidates for ceramide-regulated

enzymes [57]. Increased intracellular ceramide levels have

been involved in the activation of photoreceptor apoptosis

[58]. Furthermore, it has been suggested that LPP2 and

LPP3 play an important role in apoptotic processes. This is

supported by the fact that DAG and sphingosine, the

products of LPPs, are involved in the apoptosis induction

[39] while S1P and LPA have anti-apoptotic roles [59].

Our results regarding basal LPP activities under dark

and light conditions showed no differences between entire

ROS and ROS depleted of soluble and peripheral proteins,

thus corroborating the fact that LPPs are membrane-asso-

ciated proteins. On the contrary, it has been reported that

DAGK exhibits a different activity in ROS depleted of

soluble and peripheral proteins with respect to intact ROS

[60]. The effects of LPP alternative substrates obtained in

depleted ROS with respect to that observed in entire ROS

could be indicating the involvement of soluble and/or

peripheral proteins in the regulation of LPPs (Fig. 4). The

presence of sphingosine and ceramide strongly inhibited

LPPs activities in depleted DROS, being the activity levels

similar to those found in depleted BLROS (in the absence

of sphingosine or ceramide) (Fig. 5C).

Our findings indicate that the competition between PA

and LPA, or S1P or C1P by the active site of LPPs is

modulated by ROS illumination state and by ROS protein

association/dissociation. The role of LPPs in signaling

events and the fact that the products generated by these

enzymes control the cell-death decisions in photoreceptor

cells, open an interesting field to further studies about the

regulation of LPPs by light in these cells.

Acknowledgments This work was supported by the Consejo Nac-

ional de Investigaciones Cientıficas y Tecnicas (CONICET), the

Agencia Nacional de Promocion Cientıfica y Tecnologica (FONCyT)

and the Secretarıa General de Ciencia y Tecnologıa, Universidad

Nacional del Sur, Argentina. N.M.Giusto and G.A. Salvador are

research members of CONICET.

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